Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
A variety of cell-based assays are of considerable commercial relevance in screening for modulators of cell-based activity. For example, compounds which affect cell death can have profound biological activities and are desirably screened for in cell-based assays. Cell death has become recognized as a physiological process important in normal development, hormonal regulation of various tissues, and, e.g., in regulation of the receptor repertoires of both T and B lymphocytes. The finding that a pattern of morphological changes is common to many examples of programmed cell death (or PCD) led to the suggestion of a common mechanism, and the term xe2x80x9capoptosisxe2x80x9d was defined to include both the morphological features and the mechanism common to such programmed cell death (Kerr et al., Br. J. Cancer 26:239). This concept was extended by the finding that nuclear DNA fragmentation correlates well with apoptotic morphology (Arends et al., Am. J. Pathol. 136:593 (1990)), and the scientific literature contains many examples of PCD accompanied by these features. There are also clear examples of PCD in the absence of apoptotic morphology or DNA fragmentation (Clarke, Anat. Embryl. 181:195 (1990), Martin et al, J. Cell Biol. 106:829 (1988), and Ishigami et al., J. Immunol. 148:360 (1992)).
Cell-based assay systems model relevant biological phenomena, and have generally been widely adopted as screening assays, e.g., when screening for a compound""s effect(s) on apoptosis or other biological phenomena. Pioneering technology providing cell- and other particle-based microscale assays are set forth in Parce et al. xe2x80x9cHigh Throughput Screening Assay Systems in Microscale Fluidic Devicesxe2x80x9d WO 98/00231; in PCT/US00/04522, filed Feb. 22, 2000, entitled xe2x80x9cManipulation of Microparticles In Microfluidic Systems,xe2x80x9d by Mehta et al.; and in PCTUS00/04486, filed Feb. 22, 2000, entitled xe2x80x9cDevices and Systems for Sequencing by Synthesis,xe2x80x9d by Mehta et al.
Other cell-based assays include various methods for the preparative or analytic sorting of different types of cells. For example, cell panning generally involves attaching an appropriate antibody or other cell-specific reagent to a solid support and then exposing the solid support to a heterogeneous cell sample. Cells possessing, e.g., the corresponding membrane-bound antigen will bind to the support, leaving those lacking the appropriate antigenic determinant to be washed away. Other well-known sorting methods include those using fluorescence-activated cell sorters (xe2x80x9cFACSsxe2x80x9d). FACSs for use in sorting cells and certain subcellular components such as molecules of DNA have been proposed in, e.g., Fu, A. Y. et al. (1999) xe2x80x9cA Microfabricated Fluorescence-Activated Cell Sorter,xe2x80x9d Nat. Biotechnol. 17:1109-1111; Unger, M., et al. (1999) xe2x80x9cSingle Molecule Fluorescence Observed with Mercury Lamp Ilumination,xe2x80x9d Biotechniques 27:1008-1013; and Chou, H. P. et al. (1999) xe2x80x9cA Microfabricated Device for Sizing and Sorting DNA Molecules,xe2x80x9d Proc. Nat""l. Acad. Sci. 96:11-13. These sorting techniques utilizing generally involve focusing cells or other particles by flow channel geometry.
While cell-based assays are generally preferred in certain microscale screening applications, certain of these assays are difficult to adapt to conventional notions of high-throughput or ultra high-throughput screening assay systems. For example, one difficulty in flowing assay systems is that, during pressure-based flow of fluids in channels, non-uniform flow velocities are experienced. Faster fluid and material flow is observed in the center of a moving fluid stream than on the edge of a moving fluid stream. This non-uniform flow velocity reduces throughput for flowing assays, because assay runs have to be spaced well apart in the fluid stream to prevent overlap of materials moving at different velocities.
Accordingly, it would be advantageous to provide mechanisms for facilitating cell-based assays, including cell sorting techniques, especially in microscale systems. Additional microscale assays directed at subcellular components, such as nucleic acids would also be desirable. The present invention provides these and other features which will become clear upon consideration of the following.
The present invention relates to methods of focusing particles in microchannels, e.g., to improve assay throughput, to sort particles, to count particles, or the like. In the methods of the invention, cells and other particles are focused in the center of, to one side of, or in other selected regions of microscale channels, thereby avoiding, e.g., the above noted difficulties inherent in pressure-based flow of particles. Furthermore, the device structures of the present invention are optionally integrated with other microfluidic systems. Other reactions or manipulations involving cells, other particles, or fluids upstream of the detection zone are also optionally performed, e.g., monitoring drug interactions with cells or other particles.
In one aspect, the invention provides methods of providing substantially uniform flow velocity to particles flowing in a first microchannel. In the methods, the particles are optionally flowed in the microchannel, e.g., using pressure-based flow, in which the particles flow with a substantially non-uniform flow velocity. Prior to performing the flowing step, the particles are optionally sampled with at least one capillary element, e.g., by dipping the capillary element into a well containing the particles on a microwell plate and drawing the particles into, e.g., reservoirs, microchannels, or other chambers of the device. The particles (e.g., a cell, a set of cells, a microbead, a set of microbeads, a functionalized microbead, a set of functionalized microbeads, a molecule, a set of molecules, etc.) are optionally focused horizontally and/or vertically in the first microchannel to provide substantially uniform flow velocity to the particles in the first microchannel. Particles are optionally focused using one or more fluid direction components (e.g., a fluid pressure force modulator an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, or the like). Additional options include sorting, detecting or otherwise manipulating the focused particles.
The particles are horizontally focused in the microchannel, e.g., by introducing a low density fluid and a high density fluid into the microchannel, causing the particles to be focused in an intermediate density fluid present between the high density fluid and the low density fluid. The particles are also optionally focused in a top or a bottom portion of the microchannel by introducing a high or a low density fluid into the microchannel with the flowing particles. The particles are vertically or horizontally focused in the microchannel, e.g., by simultaneously introducing fluid flow from two opposing microchannels into the first microchannel during flow of the particles in the first channel. Vertical focusing is also optionally achieved to one side of a microchannel by simultaneously introducing fluid flow from, e.g., a second microchannel into the first microchannel during flow of the particles in the first microchannel.
In another aspect, the invention also provides particle washing or exchange techniques. For example, focused cells or other particles are optionally washed free of diffusible material by introducing a diluent into the first microchannel from at least a second channel and removing the resulting diluted diffused product comprising diluent mixed with the diffusible material through at least a third microchannel.
Alternating arrangements of diluent input and diffused product output channels are also optionally used to further wash the particles. For example, in one aspect the methods of the invention include simultaneously introducing the diluent into the first microchannel from the second microchannel and a fourth microchannel, where the second and fourth microchannel intersect the first microchannel at a common intersection region. Optionally, the methods include sequentially introducing the diluent into the first microchannel from the second microchannel and a fourth microchannel, wherein the second and fourth microchannels intersect the first microchannel at an offset intersection region. The diffused product is typically removed through the third microchannel and a fifth microchannel, which third and fifth microchannels intersect the first microchannel at a common intersection region. In further washing steps, the diluent is introduced through sixth and seventh microchannels which intersect the first microchannel at a common intersection. The resulting further diluted diffused product is removed through eighth and ninth microchannels, which intersect the first microchannel at a common intersection. Diluent is optionally introduced into the first microchannel by pressure or electrokinetic flow.
In one preferred assay of the invention, the particles are cells and the method includes performing a TUNEL assay or an Annexin-V assay on the cells in the channel to measure apoptosis.
Integrated systems for performing the above methods, including the particle sorting embodiments, are also provided.
An integrated system for providing substantially uniform flow velocity to flowing members of at least one particle population in a microfluidic device optionally includes a body structure that includes at least a first microchannel disposed therein. A first fluid direction component (e.g., a fluid pressure force modulator) is typically coupled to the first microchannel for inducing flow of a fluidic material that includes the members of the at least one particle population in the first microchannel. The first fluid direction component generally induces non-uniform flow. A source of at least one fluidic material is optionally fluidly coupled to the first microchannel. The system also optionally includes at least a second microchannel that intersects the first microchannel for introducing at least one fluid into the first microchannel to horizontally or vertically focus the members of the at least one particle population in the first microchannel. The at least one fluid is optionally introduced using a second fluid direction component that includes one or more of a fluid pressure force modulator, an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, or the like. At least one flow control regulator for regulating flow of the fluidic material or the fluid in the first or second microchannel is also optionally provided. A computer including an instruction set directing simultaneous flow of the members of the at least one particle population in the first microchannel and simultaneous introduction of the at least one fluid from the second microchannel into the first microchannel is optionally also operably coupled to a fluid movement system for directing flow of materials in the microchannels.
As a further option, this integrated system additionally includes at least a third microchannel which intersects the first microchannel in an intersection region common to the second microchannel. The flow control regulator of this system optionally further regulates flow of the at least one fluid in the second and the third microchannels. In this embodiment, the computer typically also includes an instruction set for simultaneously flowing fluids from the second and third microchannels into the first microchannel.
In particle washing systems, typically, at least fourth and fifth channels which intersect the first microchannel at a common intersection downstream of the second and third microchannels are provided. The computer further includes an instruction set for simultaneously flowing material from the first microchannel into the fourth and fifth microchannels. Sixth and seventh microchannels which intersect the first microchannel at a common intersection downstream of the fourth and fifth microchannels, with the computer further comprising an instruction set for simultaneously flowing material from the sixth and seventh microchannels into the first microchannel are optionally provided. Similarly, eighth and ninth microchannels which intersect the first microchannel at a common intersection downstream of the sixth and seventh microchannels, the computer further including an instruction set for simultaneously flowing material from the first microchannel into the eighth and ninth microchannels are optionally provided.
The integrated system optionally includes sources for any reagent or particle used in the methods noted above, such as one or more sources of terminal deoxynucleotide transferase, one or more sources of one or more fluorescein labeled nucleotides or other labeled polynucleotides, one or more sources of Annexin V, one or more sources of an AnnexinV-biotin conjugate, one or more sources of a DNA dye, one or more sources of Campthotecin, one or more sources of Calcein-AM, one or more sources of a control cell, one or more sources of a test cell, etc.
Signal detector(s) mounted proximal to the first microchannel for detecting a detectable signal produced by one or more of the members of the at least one particle population in the microchannel are typically provided in the integrated systems of the invention. The detector also optionally includes, e.g., a fluorescent excitation source and a fluorescent emission detection element. Optionally, the computer is operably linked to the signal detector and has an instruction set for converting detected signal information into digital data.
The integrated system of the present invention is also optionally used to sort the members of a particle population (e.g., a cell, a set of cells, a microbead, a set of microbeads, a functionalized microbead, a set of functionalized microbe ads, a molecule, a set of molecules, or the like). In this embodiment, the integrated system typically additionally includes a third and a fourth microchannel which intersect the first microchannel downstream from the intersection of the second microchannel with the first microchannel. The fourth microchannel also generally intersects the first microchannel downstream from the intersection of the third microchannel with the first microchannel. The flow control regulator of this system optionally further regulates flow of the at least one fluid in the third or the fourth microchannels. Furthermore, the signal detector typically detects a detectable signal produced by a selected member of the particle population between the intersections of the second and the third microchannels with the first microchannel.
In this particle sorting embodiment, the computer is optionally operably linked to the first or other fluid direction component(s), the flow control regulator, and the signal detector. Additionally, the instruction set typically directs simultaneous introduction of the at least one fluid from the third microchannel into the first microchannel to horizontally or vertically focus the selected member of the particle population such that the selected member is directed into the fourth microchannel in response to the detectable signal produced by the selected member. Optionally, the instruction set further directs simultaneous introduction of the at least one fluid from the third microchannel by activating a heating element (e.g., a Joule heating electrode, a conductively coated microchannel portion, etc.) disposed within the third microchannel or a well that fluidly communicates with the third microchannel.
In another embodiment, at least a portion of the first microchannel optionally includes a separation element disposed therein. The separation element optionally includes, e.g., two sides and at least a portion of the separation element is typically disposed upstream of the fourth microchannel. In this embodiment, a selected member of the particle population is generally directed to one of the two sides of the separation element and into the fourth microchannel that intersects the first microchannel in response to the detectable signal produced by the selected member.
The integrated system for use in particle sorting also optionally includes a fifth microchannel which intersects the first microchannel in an intersection region common to the second microchannel. In this case, the flow control regulator also typically regulates flow of the at least one fluid in the second and the fifth microchannels, and the computer optionally includes an instruction set for simultaneously flowing fluids from the second and the fifth microchannels into the first microchannel. Similarly, the system also optionally includes a sixth microchannel which intersects the first microchannel in an intersection region common to the third microchannel. In this embodiment, the flow control regulator optionally additionally regulates flow of the at least one fluid in the third and the sixth microchannels. Furthermore, the computer also typically includes an instruction set for flowing fluids from the third and the sixth microchannels into the first microchannel. Optionally, the instruction set directs individual or simultaneous fluid flow from the third and sixth microchannels by individually or simultaneously activating at least one heating element (e.g., a Joule heating electrode, a conductively coated microchannel portion, or the like) disposed within each of the third and sixth microchannels or within at least one well that fluidly communicates with each of the third and sixth microchannels.
Many additional aspects of the invention will be apparent upon review, including uses of the devices and systems of the invention, methods of manufacture of the devices and systems of the invention, kits for practicing the methods of the invention and the like. For example, kits comprising any of the devices or systems set forth above, or elements thereof, in conjunction with packaging materials (e.g., containers, sealable plastic bags, etc.) and instructions for using the devices, e.g., to practice the methods herein, are also contemplated.